FAQ: Are there any differences between the requirements for 2-3 fragment
assemblies versus 4–6?

The major differences between the two are the length of overlapping sequences
between the adjacent fragments and the incubation time of the assembly reaction.
The 15 minute assembly reaction protocol is recommended for assembly of 2–3
fragments that are flanked by 15–25 nt overlaps. The 1 hour assembly protocol
is recommended for the assembly of up to 6 fragments, flanked by 20–80 nt
overlaps. The total amount of DNA in a 4–6 fragment assembly is also higher
than a 2–3 fragment assembly.